Consequences Interactions between chemical carcinogens and DNA, which result in damage to DNA, are considered a necessary, but not sufficient, early 'initiating' step in the process of chemical carcinogenesis. Our studies have concentrated on mechanisms of interaction between chemical carcinogens, some of which are commonly used drugs, and DNA. Topics under study include both the extent of DNA adduct formation and persistence, and the biological consequences of DNA damage in cultured cells, animal models and human tissues. Information on DNA adduct processing in nuclear and mitochondrial DNA are correlated with specific effects of exposure, including tumorigenesis and human cancer risk, clinical response, specific toxicities and functional alterations in target organs and organelles. Having pioneered one of the earliest methodologies for measuring DNA damage in humans, we are interested in searching out themes that are common to both animal models and human subjects. We wish to apply the knowledge gained to enhance or reduce a specific effect in humans, and to lower cancer risk through the use of chemoprevention. The carcinogens of intensive investigation include: polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants; tamoxifen (TAM), used for adjuvant chemotherapy; and the antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) used for therapy of human immunodeficiency virus type 1 (HIV-1).

Our long-term studies in the area of PAH carcinogenesis have demonstrated that DNA damage, caused by a family of carcinogenic PAHs, can be measured in a variety of human tissues by immunoassay, and localized in particular cell types by immunohistochemistry (IHC). We have shown that human blood cell PAH-DNA adducts are formed as a result of dietary ingestion of PAHs, and increase or decrease as a function of the level of ingested PAHs. In addition, we found that PAH-DNA adducts from dietary exposure have a half-life of about a week in human blood cell DNA. When PAH levels in the diet are low, inhalation is a significant source of PAH exposure, and we reported that moving the same cohort from an area with a clean ambient environment to one that was much more polluted resulted in significant increases in blood cell PAH-DNA adduct levels. Pollution may vary by season, and we found a significantly higher level of blood cell PAH-DNA adducts in Mexico City in the dry season, when ambient PAH levels were the highest, compared to the rainy season. We have shown localization of PAH-DNA adducts in all human tissues investigated, including placenta, cervix, vulva, prostate and esophagus, suggesting that PAH-DNA adducts have widespread distribution in human organs. In addition, we have found PAH-DNA adduct formation in epidermis of Gulf of Mexico whales exposed to the 2010 Deepwater Horizon oil spill, and in intestines of beluga whales from the St. Lawrence Estuary, a target tissue for intestinal cancers believed caused by PAH contamination in the Saguenay River. With respect to human cancer risk, we demonstrated a 3-fold increased risk of colorectal adenoma in individuals with the highest leukocyte PAH-DNA adducts, compared to polyp-free controls. These were, coincidentally, the individuals who consistently ate the largest quantities of the most heavily cooked beef. Taken together, our PAH studies suggest that PAH-DNA levels vary with PAH exposure, and decreasing PAH-DNA levels may decrease human cancer risk.

Cell culture studies using normal human mammary epithelial cell (NHMEC) strains have explored the interindividual metabolic variability of the PAH benzo[a]pyrene (BP) and the pathways leading to DNA adduct formation. When NHMECs were cultured from 20 different individuals and exposed to BP, a known human carcinogen, broad interindividual variability was found for formation of the predominant DNA adduct BPdG, the induction of CYP1A1 and 1B1 RNA, and induction of the combined CYP1A1/1B1 enzyme (EROD) activity. Expression of CYP1A1 and CYP1B1, as gene abundance (copy levels/ng RNA), in NHMEC strains exposed to BP showed higher basal and BP-induced RNA copy number for CYP1B1, compared to CYP1A1, however only the CYP1A1 RNA levels correlated with formation of BPdG. Furthermore, EROD assay activity correlated with BPdG level, but activity of the detoxification enzyme NQO1 did not. Therefore, interindividual differences in BP-DNA damage in NHMECs, and possibly other human cells, are primarily related to the activity of CYP1A1, and not CYP1B1.

The formation of TAM-DNA adducts in human endometrium is a controversial topic of interest, as TAM-exposed women are at risk for endometrial cancer. Our current efforts are focused on searching for evidence of a genotoxic mechanism in tissues from women and female monkeys exposed to TAM. We demonstrated the presence of TAM-DNA adducts in human endometrial tumor and normal surrounding tissue from cancer patients receiving TAM therapy, but not in corresponding endometrial tissues from cancer patients receiving no TAM. In addition, TAM-DNA damage was found in uterus of TAM-exposed monkeys but not in the unexposed animals. Therefore, TAM induction of endometrial cancer is likely to occur, at least partially, through a genotoxic mechanism.

Antiretroviral drug combinations including nucleoside reverse transcriptase inhibitors (NRTIs) are used as therapy for individuals infected with the human HIV-1. These drugs are incorporated into DNA acting as replication chain terminators. However, mitochondrial toxicities limit NRTI use, and genotoxicity studies suggest that exposure may confer a long-term cancer risk. We have hypothesized that NRTI incorporation and sequelae are fundamental to both the nuclear and mitochondrial manifestations of toxicity. We are particularly interested in HIV-1-uninfected infants born to HIV-1-infected mothers receiving antiretroviral (ARV) therapy because these children, though born with no HIV-1 infection, are vulnerable as a result of in utero exposure. The data suggest that fetal events induced by transplacental ARV drug exposure in humans may not be readily reversible.

We have reported novel manifestations of genotoxicity by the NRTI zidovudine (AZT) in cultured cells, including: arrest of cells in S-phase, centrosomal amplification, micronucleus formation and aneuploidy. Using tissues from neonatal HIV-1-uninfected infants born to HIV-1-infected mothers receiving NRTI therapy we demonstrated AZT-DNA incorporation, as well as DNA fragmentation by Comet assay, and mutagenesis by glycophorin A (GPA) assay. The mutagenesis persisted in ARV drug-exposed infants at 1 year of age. Long-term genotoxicity was also demonstrated in the patas monkey model where pregnant dams received human-equivalent NRTI protocols for the last half of gestation. Their exposed offspring, at birth, 1 year and 3 years of age all showed significantly-elevated levels of centrosomal amplification, micronuclei and aneuploidy in bone marrow stem cells, compared to the unexposed controls. It is noteworthy that a 3 year old patas is equivalent in development to a 15 year old human, demonstrating the long-term persistence of this genotoxicity.

Evidence of mitochondrial dysfunction found in children born to HIV-1-infected mothers can include clinical compromise as well as alterations in mitochondrial morphology and mitochondrial DNA (mtDNA) quantity. We have documented both mitochondrial morphological damage and mtDNA depletion in cord blood, umbilical cord and placenta from HIV-1-infected pregnant women. These end points are considered to be caused by maternal HIV-1 status and/or maternal ARV-drug use, and the relative contributions of each cannot be ascertained in clinical samples. Using the patas monkey model, in the absence of virus, we demonstrated that mitochondrial compromise found in umbilical cords from human infants exposed to NRTIs in utero was very similar to that observed in patas offspring also exposed to NRTIs in utero. Furthermore, we showed progressive and irreversible mitochondrial toxicity in liver and brain of 1 and 3 year-old patas offspring exposed to NRTIs in utero and for 6 weeks after birth. Trying several candidates we explored the potential usefulness of chemoprotective agents, and found that the stable free radical tempol protected cultured cardiomyocytes from mitochondrial toxicity induced by AZT/didanosine (ddI). Studies to examine potential protection by tempol in the patas are underway. These mitochondrial studies are particularly important because some children born to HIV-1-infected mothers have a reduction in heart left ventricle muscle mass that may have consequences later in life.

Miriam C. Poirier is Head of the Carcinogen-DNA Interactions Section at the National Cancer Institute (NIH). She received a BSc in Chemistry, from Marygrove College, (Detroit, MI), an MSc in Biochemistry, from McArdle Laboratories, University of Wisconsin, (Madison WI), and a PhD in Microbiology from Catholic University of America (Washington, DC). Dr. Poirier obtained her PhD in 1977 while also working at the NIH, and shortly afterwards pioneered the use of immunoassays, with antisera specific for carcinogen-DNA adducts, for measurement of DNA damage in human tissues. Since 1997 she has been Head of the Carcinogen-DNA Interactions Section of the Laboratory for Cancer Biology and Genetics (CCR, NCI). Dr. Poirier is a recipient of the NIH Merit Award, the Marygrove College Distinguished Alumna Award, the NCI Leading Diversity Award, the NCI WSA Mentoring and Leadership Award, and the Environmental Mutagenesis and Genomics Society Alexander Hollaender Award.

Consequences Interactions between chemical carcinogens and DNA, which result in damage to DNA, are considered a necessary, but not sufficient, early 'initiating' step in the process of chemical carcinogenesis. Our studies have concentrated on mechanisms of interaction between chemical carcinogens, some of which are commonly used drugs, and DNA. Topics under study include both the extent of DNA adduct formation and persistence, and the biological consequences of DNA damage in cultured cells, animal models and human tissues. Information on DNA adduct processing in nuclear and mitochondrial DNA are correlated with specific effects of exposure, including tumorigenesis and human cancer risk, clinical response, specific toxicities and functional alterations in target organs and organelles. Having pioneered one of the earliest methodologies for measuring DNA damage in humans, we are interested in searching out themes that are common to both animal models and human subjects. We wish to apply the knowledge gained to enhance or reduce a specific effect in humans, and to lower cancer risk through the use of chemoprevention. The carcinogens of intensive investigation include: polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants; tamoxifen (TAM), used for adjuvant chemotherapy; and the antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) used for therapy of human immunodeficiency virus type 1 (HIV-1).

Our long-term studies in the area of PAH carcinogenesis have demonstrated that DNA damage, caused by a family of carcinogenic PAHs, can be measured in a variety of human tissues by immunoassay, and localized in particular cell types by immunohistochemistry (IHC). We have shown that human blood cell PAH-DNA adducts are formed as a result of dietary ingestion of PAHs, and increase or decrease as a function of the level of ingested PAHs. In addition, we found that PAH-DNA adducts from dietary exposure have a half-life of about a week in human blood cell DNA. When PAH levels in the diet are low, inhalation is a significant source of PAH exposure, and we reported that moving the same cohort from an area with a clean ambient environment to one that was much more polluted resulted in significant increases in blood cell PAH-DNA adduct levels. Pollution may vary by season, and we found a significantly higher level of blood cell PAH-DNA adducts in Mexico City in the dry season, when ambient PAH levels were the highest, compared to the rainy season. We have shown localization of PAH-DNA adducts in all human tissues investigated, including placenta, cervix, vulva, prostate and esophagus, suggesting that PAH-DNA adducts have widespread distribution in human organs. In addition, we have found PAH-DNA adduct formation in epidermis of Gulf of Mexico whales exposed to the 2010 Deepwater Horizon oil spill, and in intestines of beluga whales from the St. Lawrence Estuary, a target tissue for intestinal cancers believed caused by PAH contamination in the Saguenay River. With respect to human cancer risk, we demonstrated a 3-fold increased risk of colorectal adenoma in individuals with the highest leukocyte PAH-DNA adducts, compared to polyp-free controls. These were, coincidentally, the individuals who consistently ate the largest quantities of the most heavily cooked beef. Taken together, our PAH studies suggest that PAH-DNA levels vary with PAH exposure, and decreasing PAH-DNA levels may decrease human cancer risk.

Cell culture studies using normal human mammary epithelial cell (NHMEC) strains have explored the interindividual metabolic variability of the PAH benzo[a]pyrene (BP) and the pathways leading to DNA adduct formation. When NHMECs were cultured from 20 different individuals and exposed to BP, a known human carcinogen, broad interindividual variability was found for formation of the predominant DNA adduct BPdG, the induction of CYP1A1 and 1B1 RNA, and induction of the combined CYP1A1/1B1 enzyme (EROD) activity. Expression of CYP1A1 and CYP1B1, as gene abundance (copy levels/ng RNA), in NHMEC strains exposed to BP showed higher basal and BP-induced RNA copy number for CYP1B1, compared to CYP1A1, however only the CYP1A1 RNA levels correlated with formation of BPdG. Furthermore, EROD assay activity correlated with BPdG level, but activity of the detoxification enzyme NQO1 did not. Therefore, interindividual differences in BP-DNA damage in NHMECs, and possibly other human cells, are primarily related to the activity of CYP1A1, and not CYP1B1.

The formation of TAM-DNA adducts in human endometrium is a controversial topic of interest, as TAM-exposed women are at risk for endometrial cancer. Our current efforts are focused on searching for evidence of a genotoxic mechanism in tissues from women and female monkeys exposed to TAM. We demonstrated the presence of TAM-DNA adducts in human endometrial tumor and normal surrounding tissue from cancer patients receiving TAM therapy, but not in corresponding endometrial tissues from cancer patients receiving no TAM. In addition, TAM-DNA damage was found in uterus of TAM-exposed monkeys but not in the unexposed animals. Therefore, TAM induction of endometrial cancer is likely to occur, at least partially, through a genotoxic mechanism.

Antiretroviral drug combinations including nucleoside reverse transcriptase inhibitors (NRTIs) are used as therapy for individuals infected with the human HIV-1. These drugs are incorporated into DNA acting as replication chain terminators. However, mitochondrial toxicities limit NRTI use, and genotoxicity studies suggest that exposure may confer a long-term cancer risk. We have hypothesized that NRTI incorporation and sequelae are fundamental to both the nuclear and mitochondrial manifestations of toxicity. We are particularly interested in HIV-1-uninfected infants born to HIV-1-infected mothers receiving antiretroviral (ARV) therapy because these children, though born with no HIV-1 infection, are vulnerable as a result of in utero exposure. The data suggest that fetal events induced by transplacental ARV drug exposure in humans may not be readily reversible.

We have reported novel manifestations of genotoxicity by the NRTI zidovudine (AZT) in cultured cells, including: arrest of cells in S-phase, centrosomal amplification, micronucleus formation and aneuploidy. Using tissues from neonatal HIV-1-uninfected infants born to HIV-1-infected mothers receiving NRTI therapy we demonstrated AZT-DNA incorporation, as well as DNA fragmentation by Comet assay, and mutagenesis by glycophorin A (GPA) assay. The mutagenesis persisted in ARV drug-exposed infants at 1 year of age. Long-term genotoxicity was also demonstrated in the patas monkey model where pregnant dams received human-equivalent NRTI protocols for the last half of gestation. Their exposed offspring, at birth, 1 year and 3 years of age all showed significantly-elevated levels of centrosomal amplification, micronuclei and aneuploidy in bone marrow stem cells, compared to the unexposed controls. It is noteworthy that a 3 year old patas is equivalent in development to a 15 year old human, demonstrating the long-term persistence of this genotoxicity.

Evidence of mitochondrial dysfunction found in children born to HIV-1-infected mothers can include clinical compromise as well as alterations in mitochondrial morphology and mitochondrial DNA (mtDNA) quantity. We have documented both mitochondrial morphological damage and mtDNA depletion in cord blood, umbilical cord and placenta from HIV-1-infected pregnant women. These end points are considered to be caused by maternal HIV-1 status and/or maternal ARV-drug use, and the relative contributions of each cannot be ascertained in clinical samples. Using the patas monkey model, in the absence of virus, we demonstrated that mitochondrial compromise found in umbilical cords from human infants exposed to NRTIs in utero was very similar to that observed in patas offspring also exposed to NRTIs in utero. Furthermore, we showed progressive and irreversible mitochondrial toxicity in liver and brain of 1 and 3 year-old patas offspring exposed to NRTIs in utero and for 6 weeks after birth. Trying several candidates we explored the potential usefulness of chemoprotective agents, and found that the stable free radical tempol protected cultured cardiomyocytes from mitochondrial toxicity induced by AZT/didanosine (ddI). Studies to examine potential protection by tempol in the patas are underway. These mitochondrial studies are particularly important because some children born to HIV-1-infected mothers have a reduction in heart left ventricle muscle mass that may have consequences later in life.

Miriam C. Poirier is Head of the Carcinogen-DNA Interactions Section at the National Cancer Institute (NIH). She received a BSc in Chemistry, from Marygrove College, (Detroit, MI), an MSc in Biochemistry, from McArdle Laboratories, University of Wisconsin, (Madison WI), and a PhD in Microbiology from Catholic University of America (Washington, DC). Dr. Poirier obtained her PhD in 1977 while also working at the NIH, and shortly afterwards pioneered the use of immunoassays, with antisera specific for carcinogen-DNA adducts, for measurement of DNA damage in human tissues. Since 1997 she has been Head of the Carcinogen-DNA Interactions Section of the Laboratory for Cancer Biology and Genetics (CCR, NCI). Dr. Poirier is a recipient of the NIH Merit Award, the Marygrove College Distinguished Alumna Award, the NCI Leading Diversity Award, the NCI WSA Mentoring and Leadership Award, and the Environmental Mutagenesis and Genomics Society Alexander Hollaender Award.